title: Pruning the ALS-associated protein SOD1 for in-cell NMR.
abstract: To efficiently deliver isotope-labeled proteins into mammalian cells poses a main challenge for structural and functional analysis by in-cell NMR. In this study we have employed cell-penetrating peptides (CPPs) to deliver the ALS-associated protein superoxide dismutase (SOD1) into HeLa cells. Our results show that, although full-length SOD1 cannot be efficiently internalized, a variant in which the active-site loops IV and VII have been truncated (SOD1(ΔIVΔVII)) yields high cytosolic delivery. The reason for the enhanced delivery of SOD1(ΔIVΔVII) seems to be the elimination of negatively charged side chains, which alters the net charge of the CPP-SOD1 complex from neutral to +4. The internalized SOD1(ΔIVΔVII) protein displays high-resolution in-cell NMR spectra similar to, but not identical to, those of the lysate of the cells. Spectral differences are found mainly in the dynamic β strands 4, 5, and 7, triggered by partial protonation of the His moieties of the Cu-binding site. Accordingly, SOD1(ΔIVΔVII) doubles here as an internal pH probe, revealing cytosolic acidification under the experimental treatment. Taken together, these observations show that CPP delivery, albeit inefficient at first trials, can be tuned by protein engineering to allow atomic-resolution NMR studies of specific protein structures that have evaded other in-cell NMR approaches: in this case, the structurally elusive apoSOD1 barrel implicated as precursor for misfolding in ALS.
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratories of Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden.
authors.forename: Jens
authors.initials: J
authors.lastname: Danielsson
authors.forename: Kohsuke
authors.initials: K
authors.lastname: Inomata
authors.forename: Shuhei
authors.initials: S
authors.lastname: Murayama
authors.forename: Hidehito
authors.initials: H
authors.lastname: Tochio
authors.forename: Lisa
authors.initials: L
authors.lastname: Lang
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shirakawa
authors.forename: Mikael
authors.initials: M
authors.lastname: Oliveberg
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D013482:Superoxide Dismutase